Establishment of a rapid nucleic acid detection methods for Chlamydia trachomatis,Neisseria gonorrhoeae and Ureaplasma urealyticum
Objective To establish an on-site automated detection method based on a microfluidic chip platform for the simultaneous identification of Chlamydia trachomatis(CT),Neisseria gonorrhoeae(NG),and Ureaplasma urealyticum(UU).Methods The conserved sequences of the cryptic plasmid of CT,the porA gene of NG and the 16S rRNA of UU were selected as the detection targets,and specific primers and TaqMan probes were designed.Positive cloned strains of CT,NG and UU were prepared for preliminary sensitivity testing.Developed an integrated microfluidic chip based on automatic detection equipment was developed to realize the whole process of nucleic acid extraction and purification and real-time fluorescence PCR of"sample in and result out".The sensitivity,specificity,and reproducibility of the automated detection method were evaluated using liquid in-house controls for CT,NG,and UU pathogens quantified by digital PCR.The established automatic detection method and the marketed multiplex real-time PCR detection kit were used to detect 160 clinical samples at the same time to evaluate their consistency.Results The positive cloned strains were identified by agarose gel electrophoresis and DNA sequencing and constructed correctly.The detection limits of CT,NG and UU of the established fully automated assay can reach 1 102 copies/test,and the detection range is 1 102-1 107 copies/test.The method has excellent specificity and is not interfered with Herpes simplex virus type Ⅰ/Ⅱ,adenovirus,Enterovirus type 71,C.pneumoniae,M.pneumoniae and Human immunodeficiency virus type Ⅰ.The method has good reproducibility,and the coefficient of variation is＜5％.In the comparative study of 160 clinical samples,the detection rates of CT,NG and UU by the automated detection method were 15.6％,10.6％and 35.6％,respectively.There was no significant difference between the automatic detection method and the comparison kit(P＞0.05).The positive coincidence rates of CT,NG and UU were 96.2％,94.4％and 93.4％respectively,and the negative coincidence rates were 100％.Hence,the total coincidence rates were 99.4％,99.4％and 97.5％for CT,NG and UU respectively.Conclusion The established on-site automated detection method has excellent sensitivity,specificity and repeatability.This approach has obvious advantages including high degree of automation,short detection time,simple and fast operation,and is not limited by laboratory,which is suitable for clinical rapid diagnosis of CT,NG and UU.
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